The copurification of β-glucosidase, β-xylosidase, and 1,3-β-glucanase in two separate enzyme complexes isolated from Trichoderma harzianum E58

1987 ◽  
Vol 65 (9) ◽  
pp. 822-832 ◽  
Author(s):  
Larry U. L. Tan ◽  
Paul Mayers ◽  
Michelle Illing ◽  
John N. Saddler
Keyword(s):  
Isoelectric Point ◽  
Mercuric Chloride ◽  
Trichoderma Harzianum ◽  
Inhibition Constant ◽  
Activity Ratios ◽  
Molecular Masses ◽  
Inhibition Constants ◽  
Inhibition Studies ◽  
Enzyme Complexes

Two enzyme complexes, each with β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21), β-xylosidase (β-D-xylan xylohydrolase, EC 3.2.1.37), and 1,3-β-glucanase (laminarinase, EC 3.2.1.39) activity, were purified to near homogeneity from the cellulolytic fungus Trichoderma harzianum E58. The two complexes had the same isoelectric point of pH 8.3 and identical subunit molecular masses of 75 400 daltons. The two complexes were also similar in that all activities were sensitive to inhibition by mercuric chloride (2 mM) and D-glucono-1,5-lactone (0.2% w/v). The activity ratios of the major and minor complexes were 1:1.7:4.3 and 1:1.6:3.1 for the β-xylosidase, β-glucosidase, and 1,3-β-glucanase, respectively. Both complexes had approximately the same Km values for p-nitrophenyl β-D-glucopyranoside and salicin. The pH optima of corresponding activities of the two complexes were also similar. The major and minor complexes differed in that the Km of the former for laminarin was almost threefold lower than that of the latter. Whereas all three activities of the minor complexes were inhibited by D-glucono-1,5-lactone with the same inhibition constant, the β-glucosidase and 1,3-β-glucanase of the major complex had inhibition constants which differed by more than 80 000 times. In addition, the inhibition on the 1,3-β-glucanase in the major and minor complexes using D-glucono-1,5-lactone were noncompetitive and competitive, respectively. From the inhibition studies, the β-glucosidase, β-xylosidase, and 1,3-β-glucanase activities in the minor complex were deduced to be more interdependent than the same activities in the major complex.

Alkaline phosphatases of Neurospora crassa. Part II product inhibition studies

1972 ◽  
Vol 18 (4) ◽  
pp. 407-421 ◽  
Author(s):  
F. W. J. Davis ◽  
Howard Lees
Keyword(s):  
Neurospora Crassa ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Nitrophenyl Phosphate ◽  
Wide Range ◽  
Phosphate Hydrolysis ◽  
Non Linear ◽  
Inhibition Studies ◽  
Enzyme Complexes ◽  
Hydrolysis Of

A partially purified preparation of the constitutive alkaline phosphatase from Neurospora crassa, containing two electrophoretically distinct activities was used in initial studies of product inhibition patterns. Inorganic phosphate was shown to be a linear competitive inhibitor, and p-nitrophenol to be a non-linear, non-competitive inhibitor of p-nitrophenyl phosphate hydrolysis. Glycerol was shown to be a linear non-competitive inhibitor of β-glycerophosphate hydrolysis.A purification procedure whereby one enzyme activity could be obtained free of the second was devised. The purified enzyme catalyzed the hydrolysis of a wide range of substrates and had a molecular weight of 111 000. Its hydrolysis of glucose 6-phosphate was competitively inhibited by phosphate and non-competitively inhibited by glucose. Both inhibitions were linear. Hydrolysis of p-nitrophenyl phosphate was competitively inhibited by phosphate in a linear manner, but p-nitrophenol was a non-linear, non-competitive inhibitor. Alternate product inhibition by glucose was linear competitive. No inhibition by p-nitrophenol of glucose 6-phosphate hydrolysis could be detected.The inhibition data for glucose 6-phosphate and β-glycerophosphate may be consistent with an ordered Uni-Bi mechanism expanded to include one or more isomerizations of enzyme complexes. The postulation of a different mechanism involving alternate pathways is probably required to explain the data obtained when p-nitrophenyl phosphate was the substrate.

scholarly journals Purification and kinetics of phenylpropanoid O-methyltransferase activities from Brassica oleracea

1989 ◽  
Vol 67 (11-12) ◽  
pp. 763-769 ◽  
Author(s):  
Emidio De Carolis ◽  
Ragai K. Ibrahim
Keyword(s):  
Competitive Inhibition ◽  
Divalent Cations ◽  
Product Inhibition ◽  
Affinity Column ◽  
Ph Optimum ◽  
Substrate Interaction ◽  
Interaction Kinetics ◽  
Molecular Masses ◽  
Inhibition Studies ◽  
Kinetics Of

Two phenylpropanoid O-methyltransferase isoforms were purified to homogeneity from young cabbage leaves. They catalyzed the meta-O-methylation of caffeic and 5-hydroxyferulic acids to ferulic and sinapic acids, respectively. Both isoforms I and II exhibited different elution patterns from a Mono Q column, distinct apparent pIs on chromatofocusing, different product ratios, and stability on adenosine–agarose affinity column. On the other hand, both isoforms had similar apparent molecular masses (42 kilodaltons) and a pH optimum of 7.6. They exhibited no requirement for divalent cations and were both irreversibly inhibited by iodoacetate. Substrate interaction kinetics of the more stable isoform I, using the 5-hydroxyferulic acid and S-adenosyl-L-methionine, gave converging lines. Product inhibition studies showed competitive inhibition between S-adenosyl-L-methionine and S-adenosyl-L-homocysteine and non-competitive inhibition between the phenylpropanoid substrate and its methylated product. The kinetic patterns are consistent with an ordered bi bi mechanism, where S-adenosyl-L-methionine is the first substrate to bind and S-adenosyl-L-homocysteine is the last product released.Key words: phenylpropanoid O-methyltransferase, purification, isoforms, adenosine–agarose affinity chromatography, kinectic mechanism.

Kinetic Studies of Phosphoribosyl-formylglycineamidine Synthetase

1972 ◽  
Vol 50 (5) ◽  
pp. 490-500 ◽  
Author(s):  
Samuel Y. Chu ◽  
J. Frank Henderson
Keyword(s):  
Ammonium Chloride ◽  
Initial Velocity ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Free Enzyme ◽  
Ping Pong ◽  
Inhibition Constants ◽  
Inhibition Studies

Initial velocity and product inhibition studies of phosphoribosyl-formylglycineamidine synthetase indicate that the reaction involves a fully ping pong mechanism in which glutamine binds to the free enzyme and glutamate is released before the addition of ATP. ADP is released, and phosphoribosyl-formylglycineamide then binds; the liberation of Pi is rapid, and phosphoribosyl-formylglycineamidine is the last product released from the enzyme. The Km values for glutamine, ATP, and phosphoribosyl-formylglycineamide are 1.1 × 10−4 M, 1.5 × 10−3 M, and 1.1 × 10−4 M, respectively. The Km value for ammonium chloride is 7.5 × 10−3 M, and the ratio of Vmax values with ammonium chloride and glutamine is 1/40. The inhibition constants for FGAM and Pi were calculated to be 1.3 × 10−4 M and 6.45 × 10−3 M, respectively.

scholarly journals Characteristic band pattern in western blots for specific detection of anti-Trichinella spiralis antibodies in different host species

2014 ◽  
Vol 64 (1) ◽  
pp. 33-43 ◽  
Author(s):  
Nataša Ilić ◽  
Alisa Gruden-Movsesijan ◽  
Milena Živojinović ◽  
Ljiljana Sofronić-Milosavljević
Keyword(s):  
Host Species ◽  
Trichinella Spiralis ◽  
Standard Test ◽  
Antibody Detection ◽  
Reference Laboratory ◽  
Serum Samples ◽  
Western Blots ◽  
Muscle Larvae ◽  
Molecular Masses ◽  
Inhibition Studies

Abstract Western blot (Wb) is considered to be the gold standard test for Trichinella infection serology, since this method allows specific Trichinella antigens to be distinguished from cross-reactive antigens. This is not the case with widely used antibody assay techniques - indirect immunofluorescence and ELISA - which are sensitive, but subject to crossreactions that make the interpretation of weakly positive results difficult. Application of Trichinella spiralis muscle larvae excretory-secretory (ES) antigens for the specific antibody detection in ELISA resulted in improved specificity compared to that of crude worm extract that was previously in use, but since production of ES has not yet been standardized, differences among laboratories occur. For this reason, the Wb profile of serum samples from different T. spiralis infected host species: human, horse, swine and dog, was investigated in the Serbian National Reference Laboratory for Trichinellosis (NRLT). The common feature of the obtained Wb profiles was the appearance of a triad of bands with molecular masses (Mw) of 45, 49, and 53 kDa. The very same triad was recognized by a monoclonal antibody (mAb) 7C2C5 specific for an immunodominant epitope unique to the muscle larvae stage of all species in the genus Trichinella. Inhibition studies confirmed that mAb and anti-Trichinella antibodies from sera competed for the same parasite epitope. Based on the obtained results, the NRLT introduced the recognition of the above mention triad as the basis for specific anti-Trichinella antibodies detection in the sera of infected hosts.

A human cytochrome P-450 characterized by inhibition studies as the sparteine–debrisoquine monooxygenase

1984 ◽  
Vol 62 (7) ◽  
pp. 860-862 ◽  
Author(s):  
T. Inaba ◽  
M. Nakano ◽  
S. V. Otton ◽  
W. A. Mahon ◽  
W. Kalow
Keyword(s):  
Correlation Coefficient ◽  
Human Liver ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Human Cytochrome ◽  
Competitive Inhibitors ◽  
Inhibition Constants ◽  
Inhibition Studies ◽  
Cytochrome P 450

The present study compares the debrisoquine monooxygenase and the sparteine monooxygenase activities of human liver microsomes. In the presence of 14 competitive inhibitors, apparent inhibition constants (Ki) as determined by these two activities ranged over four orders of magnitude with a correlation coefficient 0.99. These in vitro results represent the strongest evidence to date that the debrisoquine monooxygenase and the sparteine monooxygenase are identical and involve a single isozyme of cytochrome P-450.

A Comparison of Inhibition Constants for the Inhibition of α-Lytic Protease and Porcine Elastase by Acetylated Alanine Peptides

1973 ◽  
Vol 51 (1) ◽  
pp. 112-114 ◽  
Author(s):  
M. C. Shaw ◽  
D. R. Whitaker
Keyword(s):  
Methyl Ester ◽  
Binding Sites ◽  
Degree Of Polymerization ◽  
Inhibition Constant ◽  
Inhibition Constants

A comparison was made of the values of the inhibition constant Ki for the inhibition of α-lytic protease and porcine elastase by various N-acetylated L- and D-alanine peptides with N-benzoyl-L-alanine methyl ester as substrate for both enzymes. The Ki's for the inhibition of elastase by N-acetyl-L-alanine peptides decreased sharply as the degree of polymerization of the inhibitor increased, the respective values for monomer, dimer, and trimer being 1200 mM, 26 mM, and 2.7 mM. The corresponding estimates of Ki for α-lytic protease, viz. 310, 270, and 70 mM, showed comparatively little dependence on the degree of polymerization of the inhibitor and suggest a substantial difference in the binding sites of the two enzymes. The Ki's for inhibition of elastase by N-acetylated L-Ala-D-Ala, D-Ala-L-Ala, and D-Ala-D-Ala were, respectively, 55, 180, and > 300 mM; those for α-lytic protease were all > 300 mM.

Synthesis of 6-Aryloxy- and 6-Arylalkoxy-2-chloropurines and Their Interactions with Purine Nucleoside Phosphorylase from Escherichia coli

1999 ◽  
Vol 54 (12) ◽  
pp. 1055-1067 ◽  
Author(s):  
Agnieszka Bzowska ◽  
Lucyna Magnowska ◽  
Zygmunt Kazimierczuk
Keyword(s):  
Mixed Type ◽  
Purine Nucleoside Phosphorylase ◽  
Phase Transfer ◽  
Competitive Inhibition ◽  
Purine Nucleoside ◽  
Inhibition Constant ◽  
Purine Base ◽  
Nucleoside Phosphorylase ◽  
E Coli ◽  
Inhibition Constants

The phase transfer method was applied to perform the nucleophilic substitution of 2,6- dichloropurines by modified arylalkyl alcohol or phenols. Since under these conditions only the 6-halogen is exchanged, this method gives 2-chloro-6-aryloxy- and 2-chloro-6-arylalkoxypurines. 2-Chloro-6-benzylthiopurine was synthesized by alkylation of 2-chloro-6-thiopurine with benzyl bromide. The stereoisomers of 2-chloro-6-(1-phenyl-1-ethoxy)purine were obtained from R- and S-enantiomers of sec.-phenylethylalcohol and 2,6-dichloropurine. All derivatives were tested for inhibition with purified hexameric E. coli purine nucleoside phosphorylase (PNP). For analogues showing IC50 < 10 μm, the type of inhibition and inhibition constants were determined. In all cases the experimental data were best described by the mixed-type inhibition model and the uncompetitive inhibition constant, Kiu, was found to be several-fold lower than the competitive inhibition constant, Kic. This effect seems to be due to the 6-aryloxy- or 6-arylalkoxy substituent, because a natural PNP substrate adenine, as well as 2-chloroadenine, show mixed type inhibition with almost the same inhibition constants Kiu and KiC. The most potent inhibition was observed for 6-benzylthio-2-chloro-, 6-benzyloxy-2-chloro-, 2-chloro-6-(2-phenyl-l-ethoxy), 2-chloro-6-(3-phenyl-l-propoxy)- and 2-chloro-6-ethoxypurines (Kiu = 0.4, 0.6, 1.4, 1.4 and 2.2 μm, respectively). The R-stereoisomer of 2-chloro-6-(1pheny-1-ethoxy)purine has Kiu = 2.0 μm, whereas inhibition of its S counterpart is rather weak (IC50> 12 μm). More rigid (e.g. phenoxy-), non-planar (cyclohexyloxy-), or more bulky (2,4,6-trimethylphenoxy-) substituents at position 6 of the purine base gave less potent inhibitors (IC50 = 26, 56 and >100 μm, respectively). The derivatives are selective inhibitors of hexameric “high-molecular mass” PNPs because no inhibitory activity vs. trimeric Cellulomonas sp. PNP was detected. By establishing the ligand-dependent stabilization pattern of the E. coli PNP it was shown that the new derivatives, similarly as the natural purine bases, are able to form a dead-end ternary complex with the enzyme and orthophosphate. It was also shown that the derivatives are substrates in the reverse synthetic direction catalyzed by E. coli PNP

scholarly journals A quick method for the determination of inhibition constants

1982 ◽  
Vol 205 (3) ◽  
pp. 631-633 ◽  
Author(s):  
S G Waley
Keyword(s):  
Competitive Inhibition ◽  
Inhibition Constant ◽  
Quick Method ◽  
Mixed Inhibition ◽  
The Common ◽  
Inhibition Constants ◽  
The Difference ◽  
The Times ◽  
Substrate Concentrations

The inhibition constant Ki in the common case of competitive inhibition can be obtained by simple comparison of progress curves in the presence and in the absence of inhibitor. The difference between the times taken for the concentration of substrate to fall to the same value is used to obtain Ki. The procedure to use when the product inhibits is described. When there is mixed inhibition, reactions at different substrate concentrations are used to obtain both inhibition constants.

scholarly journals A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors (Short Communication)

1974 ◽  
Vol 137 (1) ◽  
pp. 143-144 ◽  
Author(s):  
Athel Cornish-Bowden
Keyword(s):  
Short Communication ◽  
Graphical Method ◽  
Competitive Inhibitor ◽  
Inhibition Constant ◽  
New Method ◽  
Competitive Inhibitors ◽  
Inhibition Constants ◽  
Inhibited Enzyme

A new method is described for plotting kinetic results for inhibited enzyme-catalysed reactions. It provides a simple way of determining the inhibition constant, K′i, of an uncompetitive, mixed or non-competitive inhibitor.

Isolation of ubiquitin–E2 (ubiquitin-conjugating enzyme) complexes from erythroleukaemia cells using immunoaffinity techniques

2001 ◽  
Vol 356 (1) ◽  
pp. 199-206
Author(s):  
Koji TAKADA ◽  
Tae HIRAKAWA ◽  
Hideyoshi YOKOSAWA ◽  
Yutaka OKAWA ◽  
Hideki TAGUCHI ◽  
...  
Keyword(s):  
Heat Shock ◽  
Gel Filtration ◽  
Simple Method ◽  
Ubiquitinated Proteins ◽  
Ubiquitin System ◽  
Ubiquitin Conjugating Enzyme ◽  
Molecular Masses ◽  
Ubiquitin Molecule ◽  
E2 Enzymes ◽  
Enzyme Complexes

A variety of ubiquitin-associated (or conjugated) proteins, including substrates and enzymes for the ubiquitin system, are present in eukaryotic cells. In the present study we developed a simple method for their isolation, consisting of immunoaffinity chromatography using the monoclonal antibody FK2, which recognizes the conjugated ubiquitin molecule. Using this method followed by gel filtration, we isolated multi-ubiquitinated proteins with high molecular masses (> 30kDa) and also ubiquitinthioester-linked and mono-ubiquitinated forms of ubiquitin-conjugating (E2) enzymes, UbcH7 and UBE2N, together with mono-, di- and tri-ubiquitin molecules, from the cytoplasmic extract of heat-shock-treated K562 erythroleukaemia cells. We also demonstrated that the FK2 antibody was capable of precipitating a ubiquitin–UbcH7 thioester, but not free UbcH7, which enabled the measurement of the respective cellular levels separately. The immunoprecipitable ubiquitin–UbcH7 thioester was found only when the cells were treated with heat-shock. These results suggest the usefulness of the immunoaffinity techniques for identifying and analysing the cellular enzyme/protein–ubiquitin complexes.

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